Spargers comprising perforated plates made of nonporous materials such as ceramic or stainless steel have been used to introduce a gas in the form of bubbles into a liquid for the purpose of providing oxygen, carbon dioxide or some other gas or combination of gases to the liquid. Such spargers have a limited range of pore sizes and consequently bubble size and are relatively expensive to manufacture. Other spargers known in the art are made from materials such as sintered open-pore plastics, resin matrices enclosing spherical particles of substantially uniform size and sintered powdered metals. Due to the nature of the materials comprising these spargers it is difficult to achieve uniform bubbling and to control the size of bubbles. This lack of control may have an adverse effect upon the liquid being aerated or on biological material contained therein. For example, larger bubbles have greater shear force and may damage fragile cells in the liquid or, if the liquid is blood, cause clotting. Spargers comprised of metals may react with the liquid medium or be toxic to cells contained therein.
The sparger of the present invention represents an improvement over the prior art in that it is free of the foregoing disadvantages and inexpensive to manufacture. The material through which the gas is passed to form bubbles produces uniform bubbling and the pore size of the material is selected from a wide range to produce bubbles of the desired size. This invention represents a further improvement in that the bubbles are released into the medium in such a way as to minimize coalescing of the bubbles.
Although the sparger of the present invention may be used in any process where gas is injected into a liquid it has particular application in the field of biotechnology for the culturing of living cells, e.g., in fermentation and especially in the cultivation of eukaryotic cells such as hybridomas.
All living cells must be provided with a suitable environment in which to grow and an adequate supply of oxygen and nutrients to grow. A liquid growth medium provides for the nutritional requirements of living cells. In order to make the nutrients and oxygen available to the cells, the medium must be thoroughly mixed.
A variety of techniques and apparatus has been developed to grow cells, the simplest of which involve roller bottles containing cells and medium. The cells attach to the inside surface of the roller bottle and the bottle is rotated to provide alternate, constant bathing of the cells with liquid medium and exposure to air. The operation of more sophisticated apparatus typically involves introducing gas into the system to provide the oxygen needed for aerobic growth and circulation of the medium by internal or external mechanical means to make the nutrients in the medium and introduced gas available to the cells. The velocity at which the liquid medium circulates has an effect on cell growth. A high velocity of circulation physically stresses the cells and may damage or destroy certain types of cells. Cells of higher eukaryotes are very suceptible to damage of this type.
The circulation of the medium may be imparted in certain cell culture apparatus by introducing gas into a culture vessel in such a manner to cause circulation of the liquid medium contained therein. A vessel in which circulation of the medium is achieved in this manner typically is provided with one or more baffles separating the vessel into two or more zones through which the medium may circulate. The circulation of the medium is achieved by introducing a gas in the form of bubbles to modify the density of the liquid, i.e. reducing the density, so that the liquid will rise in the zone in which gas enters and descend in another region after losing some of the injected gas. See e.g. U.S. Pat. Nos. 3,963,581 and 4,183,787, 3,790,141 and 3,880,716. Spargers of various designs have been found useful to introduce gas into a liquid in order to impart a circulatory motion to the liquid. However, most such spargers are not useful for the cultivation of highly sensitive biological materials and particularly eukaryotic cells. Equipment designed to impart a circulatory motion to medium in the cultivation of mammalian cells has utilized perforated plates. See. U.S. Pat. No. 4,173,516.
To avoid or minimize contamination, most cell culturing apparatus use sterile techniques. The medium and the culture vessel are sterilized and a pure culture consisting of a particular living cell is introduced. In order to avoid subsequent contamination, all materials entering the system, including the large amount of oxygen required for aerobic cell culture, are sterilized. The apparatus must be designed so that contamination may be avoided during operation.
Apparatus for growing cells may be designed for batch processing or for a constant flow, steady-state processing.
In batch processing, the culture apparatus is filled with the medium and cells to be grown and growth takes place over a period of time. During this period nutrients and oxygen are supplied to the apparatus and gases produced by the growing cells are removed. However, the cells and materials produced thereby are not removed until culturing is complete.
In constant flow, steady state systems, medium is supplied to and spent medium and product is withdrawn from the reaction vessel continuously and at volumetrically equal rates. Such systems are closer to the growth conditions for mammalian cells, i.e., mammalian cells are constantly surrounded by a circulatory system that supplies nutrients and removes wastes. Some advantages of the continuous flow, steady-state system are greater yields of cell products and ease of production.
The rapidly growing field of biotechnology has provided a means for producing new and improved products by means of genetically engineered microorganisms and fused cells known as hybridomas. The commercial success of industrial applications of recombinant DNA technology and cell fusion techniques, particularly in the pharmaceutical area, will depend upon the successful, large-scale cultivation of eukaryotic cells and hybridomas. However, cultivation of such cells and hybridomas is no simple matter and presents a number of unique problems.
The organisms which have been used most extensively in cell culture have been prokaryotes (e.g. bacteria) or simple eukaryotes (e.g. yeast). These hardy organisms, encased in a tough cell wall, are relatively easy to grow as compared with higher eukaryotic cells which are larger than most microorganisms and enclosed in a delicate plasma membrane. The techniques developed for the growth of microorganisms have limited applicability to the growth of eukaryotic cells because the aforementioned techniques damage these larger, more fragile cells.
Problems with plant cell culture are similar to those of mammalian cells. The type of plant cells that the cellift is best adapted to grow are single plant cells that multiply in suspension, such as algal cells. Additional requirements for growth of plant cells are an adequate medium to provide nutrients for plant cells and a light source for photosynthesis.
Some mammalian cells grow in suspension like microbial cells and most tumor and other transformed cells can be adapted to grow in suspension. However, most higher cells must attach to a solid surface.
Although a variety of methods has been developed to grow mammalian cells both in suspension and attached to a solid surface none has solved the problem of damage described above. The simplest of these techniques is the roller bottle technique previously described herein. There are several immobilization techniques, where non-anchorage dependent ("non-ADC") cells such as hybridomas are fixed to porous ceramic or surfaces of a hollow fiber ultrafilter. Media is then oxygenated (or aerated) and pumped over the immobilized cells. Hybridomas and other cells have been encapsulated in various gelatins or membrane-life capsules for the purpose of increasing cell density, and thereby end product yield. Hybridomas and other non-ADC cells are often grown in spinner culture vessels. This vessel is a simple container with a variety of paddles, turned by a magnetic rotating control. All of the above methods suffer the disadvantage of an insufficiently gentle means to circulate the medium and thus make the nutrients and oxygen available to the cells and, in the case of cells which grow in suspension, to maintain the cells in suspension, without damage to the cells.
Outside of the in vitro systems described above the most popular method is to grow hybridomas in vivo as an ascites tumor in mice. The fluid from the tumor, harvested from the mouse peritoneum, contains the product produced by the hybridoma, i.e., monoclonal antibodies, as well as many other materials produced by the cells of the mouse as a normal by-product of cellular metabolism. Thus, the ascites fluid must be purified to remove these other products. Its use as a human therapeutic is limited both by purification problems and scale of production.
Presently available methods of culturing mammalian cells such as hybridomas are inadequate. Sensitive cells are damaged by currently available methods and such methods do not yield cells in sufficient number or cell product in sufficient quantity. Other methods are limited by purity of products produced and the need for extensive, often difficult purification. Thus, alternative methods for growing eukaryotic cells and hybridomas are currently being sought.